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Sample GSM897415 Query DataSets for GSM897415
Status Public on Oct 14, 2012
Title Xen_StageVI_mat_15
Sample type RNA
 
Source name xenopus follicles, stage VI, mature in vitro, metaphase II, replicate 2
Organism Xenopus laevis
Characteristics tissue: stage VI ovarian follicles
Stage: mature_invitro
meiotic stage: metaphase II
development capacity: developmentally fully competent
Treatment protocol The monolayers of follicular cells (=granulosa cells) surrounding the oocytes were manually recovered after a 30-min treatment with collagenase in calcium-free OR2 buffer and washed in PBS. Cell pellets were snap-frozen in liquid nitrogen after addition of TRIzol reagent and stored at -70°C until RNA extraction.
Growth protocol Sexually mature adult Xenopus laevis females (>3 year-old) were used for sampling immature vitellogenic (stage IV) and post-vitellogenic (stage VI) ovarian follicles. Mature follicles were obtained by incubating immature post-vitellogenic follicles for 15 h at room temperature in OR2 buffer (83 mM NaCl, 2.5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 1 mM Na2HPO4, 5 mM HEPES, pH 7.4) supplemented with 40 IU/mL of human chorionic gonadotropin.
Extracted molecule total RNA
Extraction protocol Total RNA was extracted using TRIzol reagent (Invitrogen, Cergy Pontoise, France). RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
Label Cy3
Label protocol Cyanine-3 (Cy3) labeled cRNA was prepared from 0.35µg RNA using the One-Color Microarray based gene Expression kit (Quick Amp Labeling, Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
 
Hybridization protocol 1.65 µg of Cy3-labelled cRNA was fragmented at 60°C for 30 minutes in a reaction volume of 55 µl containing 25x fragmentation buffer and 10x blocking agent following the manufacturers instructions (Agilent Technologies, Santa Clara, CA) . On completion of the fragmentation reaction, 55 µl of 2x GEx Hybridization Buffer HI-RPM (Agilent provided) was added to the fragmentation mixture and hybridized to Agilent Bovine Oligo Microarray (G2519F AMADID 015066) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent).
Scan protocol Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2566AA) using one color scan setting for 1x44k array slides (Scan Area 61x21.6 mm, Scan resolution 5 µm, Dye channel is set to Green and Green PMT is set to 100%).
Description Gene expression in stade VI xenopus ovulatory follicles in which oocytes have undergone maturation (metaphase II oocytes awaiting fertilization) and are developmentally fully competent.
Data processing The scanned images were analyzed with Feature Extraction Software 10.5.1.1 (Agilent) using default parameters (protocol GE1_105_Dec08 and Grid: 015066_D_20060913) to obtain background subtracted and spatially detrended Processed Signal intensities. Poor quality features that were either saturated or non-uniform were flagged using Genespring Agilent software. Only features with a signal-to-noise ratio (SNR) of up to 2.6 and significantly different from the local background (two sided t-test) were used for further analysis. Entities were then filtered based on these flag values: at least 80% of the values in any 2 out of 3 conditions must have acceptable values. Expression values were then log2-transformed and submitted to a scale normalisation
 
Submission date Mar 19, 2012
Last update date Oct 14, 2012
Contact name Julien Bobe
E-mail(s) Julien.Bobe@rennes.inra.fr
Phone (33) 2 23 48 57 24
Organization name Institut National de la Recherche Agronomique
Lab INRA-SCRIBE
Street address Campus de Beaulieu
City Rennes
ZIP/Postal code 35000
Country France
 
Platform ID GPL11258
Series (2)
GSE36603 Transcriptional profiling of somatic cells differentiation throughout oocyte competence acquisition in the xenopus ovarian follicles.
GSE36617 Contribution of molecular actors from somatic origin to oocyte developmental competence acquisition, lessons from evolution

Data table header descriptions
ID_REF
VALUE Normalized signal intensity (log2 values) calculated by Genespring Agilent GX10.0 software

Data table
ID_REF VALUE
A_10_P023096 5.7633934
A_10_P004036 8.516607
A_10_P020015 5.681168
A_10_P003052 10.040352
A_10_P020765 5.7708616
A_10_P027413 11.658327
A_10_P002294 8.047208
A_10_P005726 4.3726797
A_10_P007681 8.40268
A_10_P002347 13.468179
A_10_P004328 11.736616
A_10_P019870 5.946684
A_10_P006541 4.7523518
A_10_P010512 5.825991
A_10_P006809 7.813787
A_10_P006014 14.611886
A_10_P001812 9.076229
A_10_P003051 10.094861
A_10_P020616 3.8573766
A_10_P017504 6.9507923

Total number of rows: 9721

Table truncated, full table size 213 Kbytes.




Supplementary file Size Download File type/resource
GSM897415_Xen_stageVI_mat_15.txt.gz 6.4 Mb (ftp)(http) TXT
Processed data included within Sample table
Processed data are available on Series record

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